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Title: PURIFICATION AND CHARACTERIZATION OF BOVINE COMPLEMENT COMPONENT C3 AND ITS CLEAVAGE PRODUCTS

Author
item DICARLO, A - U OF MARYLAND
item Paape, Max
item LILIUS, E - U OF TURKU, FINLAND
item HELLMAN, J - U OF TURKU, FINLAND

Submitted to: American Journal of Veterinary Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/11/1996
Publication Date: N/A
Citation: N/A

Interpretive Summary: Complement is a series of 20 distinct proteins found in blood that play a critical role in defense against mastitis in dairy cows. Each one of these components plays a specific role in defense against mastitis. Some components recruit leukocytes to the mammary gland, while others like C3b act as bridges between invading pathogens and leukocytes. C3b will stick to bacteria and then stick to specific receptors on the surface of special kinds of leukocytes called phagocytes. This sticking process sends a signal to the phagocyte telling it to ingest and destroy the pathogen. C3b plays a critical role in defense of the mammary gland of cows against mastitis caused by coliform organisms. The udders of dairy cows are constantly exposed and challenged by millions of coliform organisms. These organisms are especially prevalent in the bedding where cows sleep. While only 2% of all udder infections are caused by coliform organisms, they account for 50% of all the clinical cases of mastitis in a herd. Because of its important role in defense against mastitis, C3b was isolated from the blood of cows, purified and its structure defined. Now that we discovered the structure of bovine C3b, it can be synthesized and used as a safe therapeutic in treating and preventing clinical mastitis in dairy cows.

Technical Abstract: The objective of this study was to purify complement component C3 from bovine serum, characterize and analyze NH2-terminal amino acid sequences from its various cleavage products, and do cross-species homology comparisons. It was found that the C3 preparation consisted of 6 segments. The molecular masses of these pieces were as follows: 30-, 40- (2 bands), 70-, 75- and 115-kd. Via sequence comparisons, the 115-kd band was identified as the alpha chain of the C3 molecule; the 70-kd piece was identified as the intact beta chain of C3; the two 40-kd bands are believed to be located at the C-terminal portion of the alpha chain, just after the cleavage site which yields C3f. The 30-kd band is the NH2-terminal portion of the alpha chain of C3b (minus the C3a segment). Sequence analysis of each band showed a high degree of homology with human, rat, mouse and horse complement component C3, with the alpha chain giving a higher level of cross species homology than the beta chain. It would appear that the purified preparation contained intact C3, C3b and the degradation products iC3b and C3c. The C3 fragments yielded high sequence homology with other species. The C3a and C3dg segments of the protein were not detected and may have been lost during the purification or lyophilization steps.